1. Field of the Invention
This invention relates to a process for preparing the releasing hormone of luteinizing hormone (LH) and of follicle stimulating hormone (FSH) in the form of an acid addition salt, to salts thereof with pharmaceutically acceptable acids, to pharmaceutical compositions containing said LH and FSH-releasing hormone, and to intermediates obtained in said process.
LH and FSH are both gonadotrophic hormones elaborated by the pituitary gland of humans and of animals. LH together with FSH stimulates the release of estrogens from the maturing follicles in the ovary and induces the process of ovulation in the female. In the male, LH stimulates the interstitial cells and is for that reason also called interstitial cell stimulating hormone (ICSH). The follicle-stimulating hormone (FSH) induces maturation of the follicles in the ovary and together with LH, plays an important role in the cyclic phenomena in the female. FSH promotes the development of germinal cells in the testes of the male. Both LH and FSH are released from the pituitary gland by the action of LH- and FSH-releasing hormones, and there is good evidence that said releasing hormone is elaborated in the hypothalamus and reaches the pituitary gland by a neurohumoral pathway, see e.g. Schally et al., Recent Progress in Hormone Reserach 24, 497 (1968).
The LH- and FSH-releasing hormone has been isolated from pig hypothalami and its constitution elucidated by Schally et al., Biochem. Res. Commun. 43, 393 and 1334 (1971), who proposed the decapeptide structure (pyro) glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH.sub.2.
This constitution has been confirmed by syntheses (see below), and the LH- and FSH-releasing hormone may also be represented in a somewhat more modern terminology by the formula 1 EQU H-Pyr-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH.sub.2. (1.)
2. Description of Prior Art
The LH- and FSH-releasing hormone has been synthesized by Sievertsson et al., Biochem Biophys. Res. Commun. 44, 1566 (1971) by a combination of classical and solid-phase (Merrifield) methods; the same hormone has also been synthesized by Geiger et al., ibid. 45, 767 (1971) using a strictly classical method; by Matsuo et al., ibid. 45, 822 (1971) using a solid-phase method; and by Monahan et al., C. R. Acad. Sci., Ser. D, 273, No. 4,508 (1971) using a solid-phase method. In contradistinction to the processes of the references cited above the process of this invention is simpler and more efficient in giving considerably better over-all yields than any of the known procedures. It is a particular advantage of the process of this invention that it requires only a minimum of protective groups for the intermediates, especially where secondary functions are concerned. Thus, the hydroxyl group in serine does not have to be protected; the NH-groups in tryptophan and in histidine do not require protection; and no protection for the guanidino function in arginine and for the hydroxyl group of tyrosine is necessary in the later stages of the process. The process of this invention is thus also more convenient and less cumbersome than the processes of Prior Art. An added advantage of the process of this invention is the fact that the final step thereof consists in the condensation of two unprotected fragments, each of which is well defined, easy to purify, and each obtainable in a high state of purity. The final product thus obtained is the free, unprotected decapeptide which does not require any deprotective steps and is obtained in a high degree of purity and in good yields.
In the following text, the term "lower alkyl" designates a straight or branched chain alkyl group containing from 1-6 carbon atoms and includes methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, t-butyl and the like. The term "lower" indicates 1-6 carbon atoms. The term "strong organic base" denotes aliphatic and heterocyclic tertiary nitrogen bases and includes triethylamine, dibutylmethylamine, N-methylpyrrolidine, N-methylpiperidine, N-methylpiperazine, N-methylmorpholine and the like; triethylamine is preferred. The term "halogenated hydrocarbon" denotes those having from 1-2 carbon atoms and includes methylene dichloride, ethylene dichloride, chloroform and the like; chloroform is preferred. The term "strong mineral acid" when used in conjunction with an anhydrous system, denotes hydrogen chloride, hydrogen bromide, and sulfuric acid; hydrogen chloride is preferred; when used in conjunction with an aqueous system the term includes any common mineral acid.
L-Pyroglutamic acid is the lactam of L-glutamic acid and has the structure of 5-oxo-L-proline.
Many of the methods used in the syntheses of peptide linkages are commonly designated by trivial names. Thus, the "azide method" comprises the reaction of an amino acid hydrazide having a suitably protected amino group with a nitrite, usually t-butyl or isoamyl nitrite, to obtain the corresponding azide which is then reacted with an amino acid having a free amino and a suitably protected carboxylic acid group, to obtain the desired peptide.
The condensation with dicyclohexylcarbodiimide comprises the reaction of an amino acid having a suitably protected amino and a free carboxylic acid group with another amino acid having a free amino and a suitably protected carboxylic acid group; the peptide linkage is formed with elimination of the elements of water and formation of dicyclohexylurea which is easily removed from the reaction mixture. In the case where the free amino group of the second amino acid reacts only with difficulty, for example if the free amino group is secondary such as in proline, it is advantageous to add hydroxysuccinimide to the reaction to form the intermediate hydroxysuccinimide ester of the first amino acid which reacts readily with a secondary amino group to form the desired peptide linkage. In principle, this modification of the dicyclohexylcarbodiimide method involves an activation of the carboxylic acid group, and such activation is also obtained when the 4-nitrophenyl or 2,4-dinitrophenyl or 2,4,5-trichlorophenyl esters of the carboxylic acid are used instead of the free acid. Such esters are generally known as activated esters.
The protective groups used in the process of this invention, and the conventional abbreviations by which they and the common amino acids are designed, are described in Schroder and Lubke, The Peptides, Academic Press, New York and London 1965.